The blood-brain barrier keeps the neural environment different from that of the blood plasma. It depends on the specialized properties of the endothelial cells of the capillaries in the brain which are much less permeable than those supplying blood to other parts of the body. Low levels of leakage in the blood-brain barrier that may be caused by nutritional deficiencies or pharmacological agents are difficult to detect, but important. We have applied quantitative confocal microscopy as an assay for low level blood-brain barrier leakage by scanning fluorescein perfused rat's brains, collecting numerous images throughout the sliced tissue and then comparing histograms of pixel ratios (fluorescein channel / background channel) obtained from a large number of optical slices. By examining tissue from vitamin E deficient and control rat minute differences may be detected which were undetectable by other methods such as fluorescence measurements of homogenized brain tissue extracts.